Xiaomeng Wang

LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling

Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequence of inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-rich alpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growth factor-β1 (TGF-β1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGF-β accessory receptor endoglin, which, in the presence of TGF-β1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a new regulator of angiogenesis that mediates its effect by modulating TGF-β signalling.

Apelin Is Required for Non-Neovascular Remodeling in the Retina

Retinal pathologies are frequently accompanied by retinal vascular responses, including the formation of new vessels by angiogenesis (neovascularization). Pathological vascular changes may also include less well characterized traits of vascular remodeling that are non-neovascular, such as vessel pruning and the emergence of dilated and tortuous vessel phenotypes (telangiectasis). The molecular mechanisms underlying neovascular growth versus non-neovascular remodeling are poorly understood. We therefore undertook to identify novel regulators of non-neovascular remodeling in the retina by using the dystrophic Royal College of Surgeons (RCS) rat and the retinal dystrophy 1 (RD1) mouse, both of which display pronounced non-neovascular remodeling. Gene expression profiling of isolated retinal vessels from these mutant rodent models and wild-type controls revealed 60 differentially expressed genes. These included the genes for apelin (Apln) and for its receptor (Aplnr), both of which were strongly up-regulated in the mutants. Crossing RD1 mice into an Apln-null background substantially reduced vascular telangiectasia. In contrast, Apln gene deletion had no effect in two models of neovascular pathology [laser-induced choroidal neovascularization and the very low density lipoprotein receptor (Vldlr)-knockout mouse]. These findings suggest that in these models apelin has minimal effect on sprouting retinal angiogenesis, but contributes significantly to pathogenic non-neovascular remodeling.

The fetal mouse metatarsal bone explant as a model of angiogenesis

The mouse fetal metatarsal provides a unique tool for studying angiogenesis. In comparison with other commonly used in vitro or ex vivo angiogenesis assays, vessel outgrowth from mouse fetal metatarsals is more representative of sprouting angiogensis in vivo. It allows the analysis of blood vessel growth, and the mechanisms underpinning this process, in a multicellular microenvironment that drives the formation of a robust and complex vascular network in the absence of exogenous growth factors. By labeling different constituents of the vascular structure, it is possible to perform 3D rendering of the spatial interplay between different cellular components and to carry out quantitative analysis of vessel outgrowth. High-resolution imaging permits the visualization of fine structural and cellular details. As the assay involves the use of fetal tissues, it is possible to follow new blood vessel formation in genetically modified mice that are perinatally lethal. The entire process takes 9–13 d. A detailed description of how to set up and perform the assay is described here.

Molecular beacon-gold nanosensors for Leucine-rich alpha-2-glycoprotein-1 (Lrg1) detection in pathological angiogenesis

Leucine-rich alpha-2-glycoprotein-1 (Lrg1) is an emerging biomarker for angiogenesis. Its expression in ocular tissues is up-regulated in both human patients with proliferative diabetic retinopathy and rodent models of pathological angiogenesis. However, there is no existing sensor that allows visualization and monitoring of Lrg1 expression noninvasively and in real time. Herein, we report a nucleic acid-gold nanorod-based nanosensor for the noninvasive monitoring of cellular Lrg1 expression in angiogenesis. Specifically, this platform is constructed by covalently conjugating molecular beacons onto gold nanorods, which prequench the fluorophores on the molecular beacons. Upon intracellular entry and endosomal escape, the complexes interact with cellular Lrg1 mRNA through hybridization of the loop area of the molecular beacons. This complexation distances the fluorophores from nanorod and restores the prequenched fluorescence. The reliability of this platform is confirmed by examining the increased Lrg1 expression in migrating keratinocytes and the Lrg1 gene changes in different postnatal stages of mouse retinal vasculature growth in the mouse retina model.